| 摘要 |
Pestivirus bovine viral diarrhea virus (BVDV) is one of the most important pathogen in cattle. BVDV strains exist as two biotypes, cytopathogenic (cp) and noncytopathogenic (ncp), according to heir effects on tissue culture cells. It has been previously reported that cell death associated to cp BVDV in vitro is mediated by apoptosis. In contrast to the situations described for other members of the Flaviviridae family (for instance, classical swine fever virus); only cp strains of BVDV express the p80 (also designated NS3 according to internationnaly recognized nomenclature) protein in virus-infected cells in vitro, uggesting its role i n the virus-associated cytopathogenicity of the virus. In this study, experiments were conducted in order to determine whether the BVDV p80 is a key-factor able to directly induce cell apoptosis. To do so, the p80- and p80.DELTA.50 (which is the p80 deleted from the NH2-terminal 50 amino acids)-cDNA encoding sequences of BVDV NADL cp strain were cloned into AdTR5-DC-GFPq transfer vector for the generation of recombinant adenoviruses (rec-Adenovirus) from which the BVDV gene of interest could be expressed from a tetracycline-responsive promoter in a di- cistronic means coexpressing the green fluorescent protein (GFP). A549tTA cells infected in vitro with p80 or p80.DELTA.50-expressing rec-Adenovirus showed cytopathogenic changes characterized by cell rounding and detachment, and nucleus chromatin condensation: DNA fragmentati on assays (oligonucleosomal DNA ladder formation on agarose gel and TUNEL) performed o n these infected cells clearly correlated the observed cytopathogenic charnges with apoptosis. Moreover, the BVDV p80 or p80.DELTA.50-induced apoptosis process correlated with the activation of cellular proteases of the ICE family (caspases), as determined by cleavage of the dea th substrate poly(ADP-ribose) polymerase (PARP). The results have also indicated that the BVDV p80.DELTA.50 appears to be a better apoptosis induces than the whole BUDV p80 as determin ed by the kinetics of PARP cleavage; quantitation of apoptotic cells over time, and by the cythopathogenic effect which appeared significantly earlier in cells infected with the BVDV p80.DELTA.50-expressing sec- Adenovirus than in cells infected with the BVDV p80-expressing sec-Adenoviru s. These results which might reflect different protein expression levels within the infected cells are consistent with the expression results obtained in bacteria with a procaryotic expressi on vector containing the p80-encoding sequence which showed no significant expression of the p80 as opposed to plasmid constructswhich readily were able to induce expression of either the p80.DELTA.26 (deletion of the NH2-terminal 26 amino acids of the p80) or p80.DELTA.50 (deletion of the NH2-terminal 50 amino acids of the p80). Finally, the results have also hown that the BVDV p80.DELTA.50-associated apoptotic process was inhibited by baculovirus p35 protein. In addition, preliminary results indicated that BVDV p80 and/or p80.DELTA.50-induced apoptosis occurs at and/ or before S phase of the cell cycle. This study constitutes the first exprimental proof that the p80 of BVDV is an induces of cell apoptosis in vitro. The results also identified the BVDV p80.DELTA.50 as a potent and powerful induces of apoptosis.
|
